BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration

Abstract

Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Talpha1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors. In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Talpha1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and Talpha1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and Talpha1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.

Fig. 1.

Expression of trkA, trkB, and trkC receptors in RSNs. trkA ISH signal is undetectable in axotomized as well as contralateral RSNs (a, b). Expression of full length trkB (c,d) as well as full-length noninserted trkC in RSNs (e, f) at 7 d after axotomy (c, e) and in contralateral RSNs (d, f). 700× magnification. Scale bar, 20 μm. RT-PCR for trkB (25 cycles, visualized by Southern blotting) using serial dilutions of the input cDNA (25.0, 12.5, 6.75 ng) obtained from axotomized (“a”) and contralateral (“c”) red nuclei (g). Note the decrease in trkB mRNA 7 d after axotomy seen by ISH (c,d) and RT-PCR (g). RT-PCR (30 cycles, ethidium bromide staining) for trkC isoforms in axotomized (“a”) and contralateral (“c”) red nuclei (h). Note the predominant expression of the noninserted trkC isoform (299 bp) as well as the isoforms with 14 amino acids (341 bp) and the weak expression of the isoforms with 25 (374 bp) and 35 (416 bp) amino acids insertions.

Fig. 2.

Schematic diagram showing the midbrain at the level of the red nucleus and the cervical spinal cord. The approximate insertion site of the application cannula connected to an osmotic minipump containing either vehicle alone or neurotrophins is illustrated in the coronal midbrain section. The hatched area in the spinal cord indicates the extent of transection at the cervical level (C3), which includes the rubrospinal tract.

Fig. 3.

Immunohistochemistry for NGF(a), BDNF (b), NT-3 (c), and NT-4/5 (d) infused lateral to the red nucleus. NGF immunostaining (a) reveals good tissue penetration of rhNGF covering almost an entire half of the midbrain in the coronal plane. In contrast, rhBDNF (b) diffusion is limited around the center of the application needle within ∼1 mm of the cannula. Penetration of rhNT-3 (c) and rhNT-4/5 (d) is comparable to that of rhNGF as shown by their respective immunostaining. 10× magnification. Scale bar, 1.5 mm.

Fig. 4.

Cresyl violet staining of vehicle- or neurotrophin-treated RSNs 14 d after axotomy (a, c, e) and their contralateral counterparts (b, d, f). Note the severe atrophy of vehicle-treated RSNs (a vs b). This atrophy is fully prevented in axotomized RSNs treated with BDNF (c) or NT-4/5 (e), displaying the cell profile sizes comparable to the contralateral RSNs (d, f). Note that the axotomized and neurotrophin-treated RSNs are chromatolytic. 400× magnification. Scale bar, 50 μm.

Fig. 6.

Histograms of the percentage of cells displaying GAP-43 (a) or Tα1-tubulin (c) expression in multiples of their contralateral expression level, obtained from representative animals infused with vehicle, BDNF, or NT-4/5 (marked by arrowsin b and d). The labels ofx-axis (multiples of contralateral) indicate an upper limit value of the bin category. Note the apparent shift to the right (increased expression) for both genes in the numbers of RSNs treated with BDNF or NT-4/5 compared with those treated with vehicle. Each symbol in b and d represents the mean (±SEM) of ISH signals/cell normalized to that of contralateral RSNs, i.e., expressed as multiples of contralateral derived from an individual animal. Dashed lines indicate the expression level of contralateral (=1). Note the increased expression of GAP-43 (b) as well as Tα1-tubulin (d) in the animal groups treated with BDNF or NT-4/5 compared with the vehicle or no-pump control groups.

Fig. 5.

GAP-43 and Tα1-tubulin ISH in axotomized RSNs treated with vehicle, BDNF, or NT-4/5. The FG-labeled, axotomized RSNs are visualized under fluorescent illumination, superimposed with autoradiographic silver grains representing the ISH signals in dark-field illumination. Note that only a subpopulation of axotomized RSNs display GAP-43 ISH signal with vehicle treatment (a); in contrast, the majority of axotomized RSNs treated with BDNF (b) or NT-4/5 (c) express high levels of GAP-43 mRNA. Moreover, an increase in Tα1-tubulin expression is also observed in axotomized RSNs treated with BDNF (e) or NT-4/5 (f) compared with those RSNs treated with vehicle only (d). 360× magnification. Scale bar, 40 μm.

Fig. 7.

Photomicrographs of FG-labeled RSNs regenerated into a peripheral nerve transplant obtained from an animal without treatment (a; c, marked byarrow) and of a BDNF-treated animal (b;c, marked by arrow). cshows the numbers of FG-labeled, i.e., regenerated, RSNs of individual animals without treatment (open symbols) and with BDNF treatment (filled symbols). Note a severalfold increase in the number of FG-positive neurons in BDNF-treated animals compared with the animals without treatment (p < 0.01; t test). 160× magnification. Scale bar, 100 μm.